1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the elucidation and uses of de-sentrinase (“SENP1”).
2. Description of Related Art
Sentrinization, a protein modification pathway present in all eukaryotic cells, is essential for cellular viability. The inventors have elucidated sentrin and the enzymatic mechanism required in the transfer of sentrin to its substrates (Okura et al., 1996; Kamitani et al., 1997a, b; Kamitani et al., 1998a; Kamitani et al., 1998b; Kamitani et al., 1998c; Gong et al., 1997; Gong et al., 1999). As shown in FIG. 1, sentrin is transferred through a unique. E1 complex (Aos1/Uba2) and E2 enzyme (Ubc9) to covalently modify a limited subset of cellular proteins (Gong et al., 1997; Gong et al., 1999).
Sentrin-1 (also called SUMO-1) is a protein that can covalently modify specific proteins in a manner analogous to ubiquitination (Okura et al., 1996; Kamitani et al., 1997a; Kamitani et al., 1997b; Matunis et al., 1996; Mahajan et al., 1997; Boddy et al., 1996; Hershko et al., 1998). In mammalian cells, there are three known sentrin family proteins that are expressed in all tissues and appear to have overlapping function (Kamitani et al., 1997a; Kamitani et al., 1997b; Kamitani et al., 1998a; Kamitani et al., 1998b).
It is now clear that the sentrinization pathway utilizes a unique activating-enzyme complex (Uba2/Aos1) and conjugation enzyme (Ubc9), to catalyze the modification of a subset of mammalian proteins, such as PML, Sp100, RanGAP1, RanBP2, IκBα, Cdc3, and cytomegalovirus IE1. (Matunis et al., 1996; Mahajan et al., 1997; Kamitani et al., 1998b; Kamitani et al., 1998c; Gong et al., 1997; Gong et al., 1999; Desterro et al., 1998; Muller et al., 1998; Sternsdorf et al., 1997; Saitoh et al., 1998). Other sentrinized proteins include Dorsal, GLUT1, GLUT4, HIPK2, p53, topoisomerase I and II, Werner syndrome gene product, MDM2, and bovine papilloma virus E1 (Yeh et al., 2000; Mao et al., 2000a; Mao et al., 2000b; Buschmann et al., 2000; Ramgasamy et al., 2000). Demonstration of sentrin modification in early studies was complicated by the presence of enzymes that cleaved the isopeptide linkage between sentrin and various target proteins in the cell lysate (Kamitani et al., 1997a; Kamitani et al., 1997b; Matunis et al., 1996). Thus, in analogy to the ubiquitin pathway (Wilkinson, 1997), enzyme(s) capable of removing sentrin from sentrinized proteins must also exist.
A Saccharomyces cerevisiae enzyme, Ulp1, was recently been shown to have de-conjugating activity for Smt-3 (yeast homologue of sentrin)-conjugated proteins and is required for cell cycle progression. Ulp1 was reported by Li et al., who showed that Ulp1 can remove Smt-3, the yeast homologue of sentrin-1, from its conjugates (Li and Hochstrasser, 1999). Ubp1 also cleaves sentrin-1/SUMO-1, but not ubiquitin, from modified proteins in vitro. Ulp1 is not related to any known de-ubiquitinating enzyme. Li et al. also cited a partial EST sequence, tentatively called HsUlp1, which is homologous to yeast Ulp1.